Urokinase plasminogen activator (uPA) is expressed in all mammalian species. It is produced by many cultured cell types of neoplastic origin and has been found more abundantly in explants of tumor tissue than in the corresponding normal tissue. uPA and its receptor, urokinase plasminogen activator receptor (uPAR), have been identified in extracts from human lung, colon, endometrial, breast, prostate and renal carcinomas, human melanomas, murine mammary tumors, the murine Lewis lung tumor, in ascites from human peritoneal carcinomatosis and human fibroblasts (Stopelli et al, Proc. Natl Acad. Sci. USA, 82:4939-43 (1985); Vassalli et al., J. Cell. Biol. 100:86-92(1985), Plow et al., J. Cell. Biol. 103:2411-2420 (1986), Boyd et al, Cancer Res., 48:3112-6 (1988); Nielsen et al., J. Biol. Chem., 263:2358-2363 (1988); Bajpai and Baker, Biochem. Biophys. Res. Commun., 133:994-1000 (1985); Needham et al., Br. J. Cancer, 55:13-16 (1987)).
uPA has been identified as the initiator of a major amplified cascade of extracellular proteolysis and/or cell migration, presumably through a breakdown of the extracellular matrix, caused by plasmin together with other proteolytic enzymes. This cascade, when regulated, is vital to certain normal physiological processes but, when dysregulated, is strongly linked to pathological processes, such as cell invasion and metastasis in cancer. Dano et al., Adv. Cancer Res. 44:139-266 (1985). There have also been reports that uPA plays a role in (1) the degradative phase of inflammation, (2) the interference of lymphocyte-mediated cytotoxicity against a variety of cells, (3) angiogenesis, (4) endothelial cell migration which is important in tumor growth, and (5) in the cytotoxic effect of natural killer cells.
uPA is a multidomain serine protease comprising (1) an N-terminal epidermal growth factor-like domain, (2) a kringle domain, and (3) a C-terminal serine protease domain. The single chain pro-uPA is activated by plasmin, which cleaves the chain into the disulfide-linked two chain active form.
The cellular receptor for uPA is uPAR, which is a multi-domain protein that is anchored by a glycolipid to the cell membrane, thus ensuring that activation of uPA is a pericellular event. Behrendt et al., Biol. Chem., 376:269-79 (1995). uPAR binds the active uPA, as well as pro-uPA and uPA bound to an inhibitor molecule DFP which binds to uPA""s active site. While the receptor binding domain of uPA has been localized to amino acids in the N-terminal growth factor-like domain region, there are varying studies which have yielded differing results regarding the span of the receptor binding domain.
For example, Stopelli et al. (Proc. Natl. Acad. Sci. USA, 82:4939-43 (1985)) first reported that the N-terminal fragment of uPA (amino acids 1-135) was sufficient for high affinity, sub-nanomolar binding to uPAR. Further work confined the uPAR binding domain to amino acids 1-48 (Robbiati et al., Fibrinolysis, 4:53-60 (1990)). Dano et al. showed that a region spanning amino acids 12-32 could block the binding of the full-length wild-type uPA to uPAR (published PCT application WO 90/12091). Other studies have shown that residues 20-30 confer the specificity of binding, but that residues 13-19 are also needed to attain the proper binding confirmation (Appella et al., J. Biol. Chem., 262:4437-40 (1987). Correspondingly, studies have shown that residues 20-30 can inhibit the binding of full-length uPA to uPAR but that a longer peptide comprising residues 17-34 is significantly more potent, requiring 10-fold less to achieve the same result. Kobayashi et al., J. Cancer, 57:727-33 (1994). Quax et al. (Arterioscler. Thromb. Vasc. Biol., 18:693-701 (1998) have reported that the receptor binding domain of uPA is localized between amino acids 20-32. In addition, Jones et al., in U.S. Pat. No. 5,942,492 have shown that cyclic peptides comprising residues 20-30 are sufficient to bind uPAR and act as antagonists of binding of uPA to uPAR and that residues N-terminal to residue 20 and C-terminal to residue 30 are not essential for high affinity binding. In view of these studies, the precise location of the uPAR high specificity binding domain in uPA is unclear.
The activity of uPA, when bound to uPAR, is confined to the cell surface by plasminogen activator inhibitors (PAI-1 and PAI-2), which bind to and inactivate the uPAR bound uPA. This tight control of uPA activity is necessary because uPA acts upon a substrate, plasminogen, that is present at a high concentration in plasma. Robbins, Meth. Ensemble., 19:184-99 (1970). uPA""s action on plasminogen produces plasmin which is a powerful broad spectrum protease that not only degrades extracellular matrix proteins directly, but also activates the latent forms of other proteases, including several metalloproteases. Werb et al., N. Eng. J. Med., 296:1017-1023 (1977); Mignatti et al., Cell, 47:487-98 (1986); He et al., Proc. Natl. Acad. Sci. USA, 86:2632-36 (1989); and Martrisian, Bioessays, 14:455-63 (1992).
In tumor biology, the link between extracelluar proteolysis and angiogenesis is clearly evident. Break-up and dissolution of existing extracellular matrix is necessary in order to create new space for blood vessels to grow into. The processes of proteolysis and angiogenesis are highly coordinated. For example, two angiogenic growth factors, basic fibroblast growth factor and vascular endothelial growth factor markedly up-regulate the production of uPA and the expression of uPAR by endothelial cells. Mignatti et al. J. Cell. Biol., 113:1193-1201 (1991); Mandriota et al, J. Biol. Chem., 270:9709-9716 (1995). Therefore, uPA and uPAR have emerged as a target for developing an anti-metastatic/anti-angiogenic therapy for cancer. Fazioli et al, Trends Pharmacological Sci., 15:25-29 (1994).
The uPA/uPAR interaction goes far beyond localizing proteolysis at the cell surface however. The mere occupation of uPAR by uPA induces, by indirect means, signal transduction events leading to one or more of the following effects: mitogenesis (Rabbani et al., J. Biol. Chem., 267:14151-56 (1992)); expression of the c-fos gene (Dumler et al., FEBS Lett., 322:37-40 (1994)); cysteine- and metalloprotease expression by macrophages (Rao et al, J. Clin. Invest., 96:465-74 (1995)); transfer of mechanical force leading to increased cytoskeletal stiffness (Wang et al., Am. J. Physiol., 268:C1062-66 (1995)); endothelial cell migration (Odekon et al., J. Cellul. Physiol., 150:258-63 (1992)); endothelial cell morphogenesis into tubular structures (Schnaper et al., J. Cellul. Physiol., 165:101-118 (1995)); and endothelial cell deformability and motility (Lu et al., FEBS Lett. 380:21-24 (1996). All of these phenomenon are blocked by blocking the access of uPA to uPAR.
In addition to binding uPA, uPAR serves as a cellular adhesion receptor for vitronectin and as a signaling receptor. Wei et al., J. Biol. Chem., 269:32380-88 (1994); Robinson, Signal transduction via GPI-anchored membrane proteins. ADP ribosylation in animal tissue. Plenum Press, NY (1997); Wei et al., J. Cell. Biol., 144:1285-1294 (1999). uPAR also interacts with several cell surface proteins including integrins, low-density lipoprotein receptor-related peptide, very-low-density lipoprotein receptor, megalin and the mannose-6-phosphate/insulin-like growth factor-II receptor. Moestrup et al., J. Biol. Chem., 268:16564-70 (1993); Heegaard et al, J. Biol. Chem., 270:20855-61 (1995); Czekay et al., Mol. Biol. Cell, 8:517-32 (1997).
uPAR is further involved in both clathrin-dependent and clathrin-independent endocytosis. Vilhardt et al., Mol. Biol. Cell, 10:179-195 (1999). Clatherin-dependent endocytosis of uPAR is believed to depend on binding of uPA:PAI-1 to uPAR and subsequent interaction with internalization receptors for the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). In contrast, clathrin independent endocytosis of uPAR, which is also believed to occur when uPA:PAI-1 is bound, is not inhibited by RAP. See Vilhardt et al., Mol. Biol. Cell, 10:179-195 (1999).
Rodenberg et al. (Biochem J., 329:55-63 (1998)) have shown that endocytosis of uPA requires both uPA and PAI-1. The complex of uPA and PAI-1 has been shown to be required for binding to the endocytosis receptors xcex12-macroglobulin receptor/low-density lipoprotein receptor-related protein (xcex12MR/LRP) and very-low-density lipoprotein receptor (VLDLR) while free uPA and PAI-1 are not able to bind to the endocytosis receptors. See Rodenberg et al., Biochem J., 329:55-63 (1998).
The region of uPA which appears to be responsible for its binding to PAI-1 has been localized to the C-terminal proteinase domain of uPA. Wei et al., J. Biol. Chem., 269:32380-8 (1994); Stoppelli et al., Proc. Natl. Acad. Sci. USA, 82:4939-43 (1985); Conese and Blasi, Biological and Chemical Hope-Seyler, 376:143-55 (1995). In addition, Rodenberg et al. have isolated a four residue region of PAI-1 which appears to be responsible for the high affinity binding of the uPA:PAI-1 complex to the endocytosis receptors. These references have suggested that endocytosis of uPA is dependent on PAI-1. Furthermore, the endocytosis of uPA has also been shown to be dependent upon uPAR. Goretzki and Mueller, J. Ce. Sci. 110:1395-402 (1997).
The effective treatment of inherited and acquired disorders through the delivery of a transgene which is capable of correcting the disorder is dependent upon the efficiency of delivery of the transgene. Various vector systems have been developed that are capable of delivering a transgene to a target cell. However, there remains a need to improve the efficiency of transgene delivery to achieve effective treatments. Improved efficiency is desirable to both increase the ability of the vector to correct the cellular defect and to decrease the toxic effects of the vector by decreasing the required amount of the vector to achieve effective treatments.
Adenovirus is a non-enveloped, nuclear DNA virus with a genome of about 36 kb. See generally, Horwitz, M. S., xe2x80x9cAdenoviridae and Their Replication,xe2x80x9d in Virology, 2nd edition, Fields et al., eds., Raven Press, New York, 1990. Recombinant adenoviruses have advantages for use as delivery systems for nucleic acid molecules coding for, inter alia, proteins, ribozymes, RNAs, antisense RNA that are foreign to the adenovirus carrier (i.e. a transgene), including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts. See Berkner, K. L., 1992, Curr. Top. Micro Immunol, 158:39-66; Jolly D., 1994, Cancer Gene Therapy, 1:51-64.
Adenoviruses have a natural tropism for respiratory tract cells which has made them attractive vectors for use in delivery of genes to respiratory tract cells. For example, adenovirus vectors have been and are being designed for use in the treatment of certain diseases, such as cystic fibrosis (CF): the most common autosomal recessive disease in Caucasians. In CF, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disturb cAMP-regulated chloride channel function, resulting in pulmonary dysfunction. The gene mutations have been found to encode altered CFTR proteins which cannot be translocated to the cell membrane for proper functioning. The CFTR gene has been introduced into adenovirus vectors to treat CF in several animal models and human patients. Particularly, studies have shown that adenovirus vectors are fully capable of delivering CFTR to airway epithelia of CF patients, as well as airway epithelia of cotton rats and primates. See e.g., Zabner et al., Nature Genetics 6:75-83 (1994); Rich et al., Human Gene Therapy 4:461-476 (1993); Zabner et al., Cell 75:207-216 (1993); Crystal et al., Nature Genetics 8:42-51 (1994).
However, it would be useful to alter the tropism of a virus, such as adenovirus, to allow it to be used to deliver a nucleic acid molecule to a variety of cells for which the virus is normally non-tropic and to improve the uptake of the virus into cells.
Certain situations exist where it would be useful to modify the tropism of viruses (such as adenovirus, adeno-associated virus, retrovirus, etc.) to target the vector to cell surface molecules other than the virus"" normal cell surface receptor. For example, certain cells are normally refractory to infection by certain viruses. It would be useful to have a method of overcoming the inability of these cells to be infected. Similarly, in cancer cells, many receptors are up-regulated, such as uPAR. Therefore, it would be useful to be able to specifically target vectors to these up-regulated receptors to increase the uptake of nucleic acids providing for antitumor agents for treatment of such cancers.
Furthermore, in practice, in order to achieve effective transfer of viral vectors into affected cells, repeated administration of the viral vector over a course of time may be required. Readministration of the viral vector can trigger an immune response within the subject to whom the vector is given, which requires subsequently higher doses of the viral vector to avoid immune elimination of the virus. If the efficiency of uptake of the viral vector is increased, a lower dose of the vector can be used, which, in turn, may help alleviate the immune response problems which are associated with the readministration of viral vectors. Similar problems may also be alleviated for other types of vectors as well, such as RNA, polynucleotides, small molecules, etc.
Administration of the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia could provide an important new treatment for cystic fibrosis (CF) lung disease. However, despite the demonstrated ability of several vectors to deliver and express CFTR and correct the Clxe2x88x92 channel defect in airway epithelia in vitro and in vivo (see O""Neal and Beaudet. Hum Mo. Genet. 3:1497-1502 (1994); Crystal. Science 270:404-10 (1995); Wilson. New England J. Med. 334:1185-7 (1996); Middleton and Alton. Thorax 53:197-9 (1998); and Welsh. J. Clin. Invest. 105:589-596 (2000), for review)), a severe inefficiency in delivering a vector coding for CFTR has impeded the clinical development of the administration of CFTR to CF patients as a new treatment. The two most important factors for the inefficiency are limited binding of vector to the apical surface of differentiated human airway epithelia and limited endocytosis across the apical membrane. A paucity of apical receptors prevents binding of viral vectors, including adenovirus (Bergelson et al. Science 275:1320-23 (1997); Pickles et al. J. Virol. 72:6014-23 (1998); Walters et al. J. Biol. Chem. 274:10219-10226(1999)), adenovirus-associated virus Teramato et al. J. Virol. 72:8904-8912 (1998); Summerford and Samulski. J. Virol. 72:1438-45 (1998); Duan et al. Hum. Gene Ther. 9:2761-76 (1998), and retroviral vectors (Wang et al. J. Virol. 72:9818-26 (1998), as well as nonviral vectors, including cationic lipids (Zabner et al. J. Biol. Chem. 270:18997-19007 (1995); Matsui et al. J. Biol. Chem. 272:1117-26 (1997); Fasbender et al. Gene Ther. 4:1173-80 (1997)). Compounding the limited vector binding, and in contrast to observations in cell lines, the rate of endocytosis across the apical membrane of differentiated airway epithelia is low and may limit administration of adenovirus, AAV, retrovirus and nonviral vectors. Pickles et al. J. Virol. 72:6014-23 (1998); Fasbender et al. Gene Ther. 4:1173-80 (1997); and Goldman and Wilson. J. Virol. 69:5951-8 (1995).
Hence, a method that increases vector binding and endocytosis may enhance both viral and non-viral vector administration to airway epithelia. One strategy to circumvent the lack of apical receptors is to non-specifically increase vector biding. For example, earlier work showed enhanced administration of adenovirus or AAV by incorporating virus in calcium phosphate (CaPi) coprecipitate. See Fasbender et al. J. Clin. Invest. 102:184-93 (1998); Lee et al. Hum. Gene Ther. 10:603-13 (1999); Walters et al. J. Virol. 74:535-540 (2000). However, this delivery method did not increase endocytosis across the apical surface. See Walters and Welsh, Gene Ther. 6:1845-1850 (1999); U.S. patent application Ser. No. 09/082,510 (incorporated herein by reference).
Other vectors comprising cationic amphiphiles such as lipids, synthetic polyamino polymers (Goldman et al., 1997, Nat. Biotechnol. 15:462-466), and poly-lysine (Kollen et al., 1996, Hum. Gene. Ther. 7:1577-1586), which are used with nucleic acid molecules, such as a plasmid, to transfect a target cell, are useful for delivery of nucleic acids to cells. Most of these vectors suffer from nonspecificity and inefficiency of delivery. Therefore, a method for targeting these systems to cells and improving the uptake of such vectors into cells would also be useful.
U.S. Pat. No.5,759,452 (""452 patent) discloses the use of an isolated portion of the A-chain of a urokinase-type plasminogen activator linked to a xe2x80x9cdrugxe2x80x9d, wherein the A-chain portion binds stably to an outer membrane of a platelet and delivers the drug to the platelet. The portion of the A-chain comprises amino acids 1 through 132 or can be the full-length urokinase-type plasminogen activator. The ""452 patent disclosure is limited to the delivery of a xe2x80x9cdrugxe2x80x9d via the urokinase-type plasminogen activator in platelets and does not provide teaching for the delivery to other cell-types, including airway epithelia, and does not provide teaching for the improved uptake of a vector (including a small molecule, protein, polynucleotide, RNA, DNA, virus, viral vector, plasmid, etc) to various cell types.
PCT Patent Application WO/9846632 discloses cyclic uPAR inhibitor peptides derived from uPA amino acids sequences 22-28 which bind to uPAR and block the binding of uPA. WO/9846632 describes the use of these peptides for the delivery of diagnostic markers or therapeutic agents to uPAR-expressing cells. WO/9846632 states that linear and cyclic peptides corresponding to amino acids 19-31 of uPA display a surprisingly higher affinity for uPAR than previously described peptides spanning amino acids 14-32 of uPA (Magdolen et al., Eur. J. Biochem. 237:743-751 (1996) and 13-19 of uPA (Appella et al., J. Biol. Chem. 262:4437-4440 (1987) which display a low affinity with uPAR. The low affinity of the peptides described by Magdolen et al. and Appella et al. are described as not being sufficient for therapeutic use creating a need for uPA peptides which feature higher affinity for the uPAR receptor.
The present invention is based in part on the unexpected finding that the seven residue peptide of uPA (corresponding to residues 13-19described by Appella et al. as a low affinity uPAR binder, is capable of high affinity binding and facilitating the delivery and endocytosis of the peptide, including the peptide linked to virus (and other cargo) by a cell bearing uPAR, even though this region of uPA does not bind PAI-1 which has previously been shown a necessary factor for the endocytosis of uPA. In addition, the present invention is also based on the previously unknown finding that airway epithelial cells express uPAR and that uPA is capable of binding thereto and facilitating endocytosis therein.
These findings have allowed for the targeted and improved delivery of adenovirus vectors to airway epithelia and is also useful for the targeted and improved delivery of cargo in general to various cell types expressing uPAR.
The present invention relates to the targeted delivery of a delivery vehicle construct which specifically binds to and stimulates endocytosis into cells expressing the urokinase plasminogen activator receptor (uPAR), and in particular, human airway epithelia. The delivery vehicle construct comprises a portion of uPA and a cargo linked thereto. The delivery vehicle construct is useful for the targeted delivery of cargo, including inter alia, small molecules, polynucleotides (coding, inter alia, for a protein, RNA, ribozyme, or antisense RNA), DNA, oligonucleotide decoys, antisense RNA, polypeptides, viruses, modified viruses, viral and non-viral vectors and plasmids, to cells expressing uPAR, and in particular, human airway epithelia. Preferred viruses, modified viruses and viral vectors are derived from adenovirus, retrovirus, herpes simplex virus, adeno-associated virus or poxvirus, with adenovirus being particularly preferred.
In one aspect of the invention, the uPA portion of the delivery vehicle construct comprises the full-length, wild-type uPA or a fragment of uPA which has the PAI-1 binding region deleted and is useful for the targeted delivery of the cargo to airway epithelia.
In a preferred aspect of the invention, the uPA portion of the vector is a seven residue peptide derived from uPA (u7-peptide) comprising amino acids 13-19 of uPA (amino acids CLNGGTC; SEQ ID NO:1, where C is Cysteine, L is Leucine, N is Asparagine, G is Glycine, and T is Threonine) and the cargo portion is an adenoviral vector, preferably containing a transgene. In a particularly preferred embodiment of the invention, the uPA portion of the vector is the u7-peptide, the cargo is an adenoviral vector containing DNA coding for CFTR and the vector is delivered to airway epithelia lacking a functional CFTR gene.
The present invention also provides a method for delivering the delivery vehicle construct to a target cell. The method comprises (a) contacting a target cell with a delivery vehicle construct comprising a uPA portion and a cargo portion; (b) delivering the cargo to the cell; and (c) producing a desired result in the cell. Such desired result may include, inter alia, the correction of a defect in the target cell (e.g. correction of a Clxe2x88x92 channel in the target cell with a transgene encoding CFTR) the destruction of the target cell (e.g. destruction of a cancerous cell with a suicide gene), or the delivery of a gene to the target cell.